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Structural basis for pathogenic variants of GJB2 and hearing levels of patients with hearing loss

Abstract

Objectives

The crystal structure of the six protomers of gap junction protein beta 2 (GJB2) enables prediction of the effect(s) of an amino acid substitution, thereby facilitating investigation of molecular pathogenesis of missense variants of GJB2. This study mainly focused on R143W variant that causes hearing loss, and investigated the relationship between amino acid substitution and 3-D structural changes in GJB2.

Methods

Patients with nonsyndromic hearing loss who appeared to have two GJB2 pathogenic variants, including the R143W variant, were investigated. Because the X-ray crystal structure of the six protomers of the GJB2 protein is known, R143W and structurally related variants of GJB2 were modeled using this crystal structure as a template. The wild-type crystal structure and the variant computer-aided model were observed and the differences in molecular interactions within the two were analyzed.

Results

The predicted structure demonstrated that the hydrogen bond between R143 and N206 was important for the stability of the protomer structure. From this prediction, R143W related N206S and N206T variants showed loss of the hydrogen bond.

Conclusion

Investigation of the genotypes and clinical data in patients carrying the R143W variant on an allele indicated that severity of hearing loss depends largely on the levels of dysfunction of the pathogenic variant on the allele, whereas a patient with the homozygous R143W variant demonstrated profound hearing loss. We concluded that these hearing impairments may be due to destabilization of the protomer structure of GJB2 caused by the R143W variant.

Peer Review reports

Introduction

GJB2 is the most frequent causative gene of autosomal recessive nonsyndromic sensorineural hearing loss (DFNB1A, OMIM: 220290) worldwide [1, 2]. GJB2 encodes connexin 26, which forms a hemichannel or connexon composed of six protomers (subunits), enabling the exchange of ions and small molecules between cells. A gap junction that allows the transport of small molecules, including K + , in the cochlear cells and is believed to be essential to maintaining high K + concentration in the cochlear endolymph via ion-recycling in the cochlea [3]. Normal ion exchange occurs when the gap junction formed by two connexons achieve the correct three-dimensional (3-D) structure.

Hearing levels of the patients having biallelic missense variants of GJB2 vary from mild to profound [4,5,6]. This could be attributed to the extent of the level of dysfunction of the GJB2 missense variants, based on the fact that a compound heterozygote with two nonsense or truncated variants generally leads to more severe phenotypes [4, 5]. Various studies have attempted to elucidate the extent of molecular pathology of GJB2 variants via in vitro analyses [7,8,9]. However, due to technical differences, the molecular pathology of some variants remains controversial [7,8,9], even though the pathogenicity of variant which change an arginine at position 143 of the GJB2 to a tryptophan (R143W) (NM_004004.6:c.427C > T) has been established clinically [10, 11].

In Ghana, R143W is the largest contributor to non-syndromic hearing impairment and has a reported prevalence of 25.9% in affected multiplex families [12].

To understand the molecular pathology of each missense variant of GJB2, predicting the structural changes in GJB2 caused by each variant is considered an alternative strategy, as it provides visual information of the structural change at the atomic level. In addition, it could provide insights for designing specific drugs to attenuate or block the dysfunctional functioning by changing the residues.

The crystal structure of the gap junction channel revealed two membrane-spanning hemichannels consisting of six GJB2 protomers [13]. Notably, R143 has been shown to interact with N206 through a hydrogen bond [13]. The two residues reside in adjacent transmembrane regions (helix-3 and helix-4) and are considered to contribute to the stability of the protomer. Interestingly, not only the R143W but also the N206S (c.617A > G) [14] and N206T (c.617A > C) [15] variants have been reported to be associated with hearing loss.

This study was aimed at predicting the structural changes in GJB2 caused by the R143W as well as the N206S and N206T variants and investigating whether the hydrogen bond between the two residues was affected. We also assessed whether the 3-D structure of GJB2 was altered by the V37I variant. Patients carrying the R143W variant in one allele and a pathogenic variant of GJB2 in the other were included, and the correlation between the genotypes and hearing levels of patients was investigated.

Methods

Molecular modeling of GJB2 variants

The R143W, N206T, N206S, and V37I variants of GJB2 were modeled on SWISS-MODEL [16, 17] using the crystal structure of GJB2 (PDB: 2zw3, A chain) [13] as a template. SWISS-MODEL is a fully automated protein homology-modeling server and one of the most widely used tools with high reliability [18]. In our previous study, the structures of its models showed high scores using the structural evaluation software Verify 3D [19,20,21]. Furthermore, to confirm the accuracy of the models, the 3-D structures corresponding to each model were predicted using Alphafold2 [22, 23], which is a novel and powerful machine learning approach with the highest reliability among the options currently available. The calculations were carried out in ColabFold v1.5.5: AlphaFold2 using MMseqs2 [24]. All diagrams were created using UCSF Chimera [25] to visualize ribbon models with the hydrogen bonds.

Subjects

Genetic testing for GJB2 variants by direct sequencing was performed on 74 individuals (46 families) with hearing loss, and 42 patients (37 families) were found to carry biallelic GJB2 variants. In total, 22 patients with nonsyndromic hearing loss who appeared to have two GJB2 pathogenic variants including at least one R143W variant were investigated. Hearing levels were examined using pure-tone audiometry, auditory steady-state response, or conditioned orientation reflex audiometry, according to the age of the patients. Hearing levels of those with better hearing were classified according to the recommendations of the Genetic Deafness study group [26]. Based on our flowchart, genetic analyses of GJB2 were performed on patients with nonsyndromic hearing loss in which nongenetic causes were excluded [27]. To predict the pathogenicity of each variant, the REVEL scoring system, an in silico pathogenicity predictor of missense variants [28], was used. Scores ≥ 0.7 were considered to be indicative of pathogenicity [29].

Results

The crystal structure of GJB2 was used as a template; then, a structural model for the R143W variant was constructed, and the resulting mechanisms of dysfunction from the viewpoint of structural biology were investigated. R143 was located in helix 3 of GJB2, and by virtue of its hydrogen bonding to N206 in helix 4, stability of the subunit structure (one unit of six protomers) was thought to be maintained (Fig. 1a, b). The hydrogen bond disappeared after substituting R143 with tryptophan (W), as shown in our structural model (Fig. 1c). Moreover, N206T and N206S have both been reported as pathogenic variants of GJB2 [14, 15, 30], which are variants of the corresponding partner residue, i.e., N206 for hydrogen bonding with R143. The REVEL scores [28] predicted the R143W as well as the N206T and N206S variants to be pathogenic (R143W; 0.918, N206T; 0.826, N206S; 0.775), supporting the findings of previous reports. Prediction of the 3-D structural models of the N206T and N206S variants revealed absence of the hydrogen bond between the side chains of the R143 and the -OH group of either the substituted T206 or S206 (Fig. 1d, e).

Fig. 1.
figure 1

3-D structures of the wild-type and variants of GJB2. a The hemichannel crystal structure consisting of six wild-type GJB2 proteins. The positions R143 and N206 in one of the GJB2 hexamers are indicated by yellow circles. b Hydrogen bond between R143 on helix 3 and N206 on helix 4 of wild-type proteins is indicated in red. ce The hydrogen bond was absent in R143W (c), N206T (d), and N206S (e) variants. Arrows indicate the position of the hydrogen bonds

Structural change in another pathogenic GJB2 variant, V37I [31], was also predicted and compared with those in the R143W, N206T, or N206S variants. When patients carried the V37I variant on an allele with the R143W or N206S variant on the other allele, they showed moderate to severe hearing loss (Additional Table S1). In the structure of GJB2, the valine at position 37 is located at the center of the pore (Additional Fig. S1). By comparing the 3-D structures of the wild and variant models, it was suggested that when the valine at position 37 was substituted with isoleucine, although isoleucine has a slightly larger side chain, no significant change was observed in the hydrophobicity of the surroundings for both wild-type and variant residues (Additional Fig. S1).

Furthermore, the hearing levels of the 34 patients carrying at least the R143W variant on an allele with known pathogenic variants on the other allele were investigated (Fig. 2). The clinical data of patients with each genotype are presented in Additional Table S1. Genotype–phenotype correlations revealed that when the other allele of R143W was a missense variant among the 11 patients harboring p.[R143W];[V37I] genotype, one patient had mild hearing loss, nine patients had moderate hearing loss, whereas one had severe hearing loss. The patient with p.[R143W];[H73Y] genotype had severe hearing loss. Among the five cases having profound hearing loss, only one case had p.[R143W];[R143W] genotype. Conversely, when the other allele included a truncated variant (frameshift or nonsense variant), profound hearing loss was observed, i.e., among 21 cases having either p.L79Cfs*3(c.235delC rs80338943), p.G45E;Y136*(rs786204690), p.H100Rfs*14 (c.299_300del, rs111033204), or p.A171Efs*40 (c.508_511dup, rs773528125) on the allele other than the R143W allele, 17 had profound hearing loss (Fig. 2). Similar to those with the R143W allele, patients with p.[N206S];[V37I] genotype showed moderate hearing loss, whereas those with p.[N206S];[truncated variants] genotypes tended to show severer hearing loss (Additional Table S1).

Fig. 2
figure 2

Hearing levels of patients carrying p.R143W variant of GJB2. Hearing levels of 22 patients with each genotype, including at least one R143W variant, were shown. See [18] for the classification of the hearing levels

Discussion

This study focused on the hydrogen bond between R143 and N206 of the adjacent helices in the GJB2 subunit (one unit of six protomers) and examined its significance. Based on the crystal structure [13] and variant models, including the pathogenic variants of R143W, N206T [15], and N206S [14, 32], the hydrogen bond was absent in all cases. This finding strongly suggests that this hydrogen bond is an indispensable component in maintaining the transmembrane domain structure of the GJB2 protomer and is essential for its proper function [33]. We also built other molecular models for three variants—namely, R143W, N206T, and N206S—using Alphafold2. As a result, it became clear that the hydrogen bonding between R143 and N206 in the wild type disappeared in all three variants, indicating the accuracy of the models made by SWISS-MODEL (Additional Fig. S2). As the position of R143 is located at a distance from the inner pore region where ion permeation occurs (even the nearest Ile 9 residue is at least 16.50 Å away), the R143W variant would have little direct effect from electrostatic interactions with potassium ions moving through the pore. Hence, the R143W variant, rather than directly affecting ion permeability, was predicted to destabilize the 3-D structure of each protomer (one unit of six), subsequently resulting in instability of the whole hemichannel (connexon) structure. This instability would also result in structural changes in the whole gap junction structure, consequently decreasing ion permeability indirectly. If the instability of the protomer with the R143W variant leads to loss of function, the level of hearing loss could be primarily determined by the level of residual function of the GJB2 protomer from the other allele. In this study, profound hearing loss was detected in patients with the p.[R143W];[R143W] genotype. Moreover, previous studies have also documented severe to profound hearing loss in all patients with the p.[R143W];[R143W] genotype [10, 11, 34]. Extremely reduced hearing levels in patients with compound heterozygotes of R143W with either a frameshift or a nonsense variant have also been observed. In case of patients with frameshift variants, patients with the p.[R143W];[G12Vfs*2 (c.35delG, rs80338939)] genotype showed profound hearing loss [5]. This finding on profound hearing loss is consistent with the prediction that the presence of the R143W variant results in severe hearing loss through the complete elimination of the gap junction function. Young patients with the p.[R143W];[L79Cfs*3] compound heterozygous variant often have profound hearing loss (75%), but relatively severe (12.5%) or moderate hearing loss (12.5%) is also observed (Additional Table S1). Childhood hearing impairment with GJB2 variations involves all frequencies and is of variable severity [35]. The variation in hearing loss severity could not be satisfactorily explained only from the view point of structural biology. More age-specific hearing data from patients with the same variant will be needed to clarify the age-specific effects of p.[R143W];[L79Cfs*3] on hearing levels.

Unlike the R143W variant that showed structural instability, only minor structural and physical–chemical changes were observed in the V37I variant when compared with the wild type. Contrary to the findings in patients with the R143W homozygote or p.[R143W];[truncating variant] genotypes, our finding that none of the patients with the p.[R143W];[V37I] genotype showed profound hearing loss is consistent with the results of previous reports [31, 34,35,36,37,38]. These results imply that the V37I variant does not significantly affect the stability of the connexon structure. Further, no significant changes in the interactions (including hydrophobic interactions) with the surrounding residues were observed when valine 37 was substituted with isoleucine. Recently, a Chinese group showed that V37I does not affect connexon formation, but causes the aggregation of detached inner wall N-terminal “plugs” and reduces channel ion flow, as revealed by molecular dynamics (MD) simulations [39]. This report suggests that the V37I variant does not affect connexon formation, but is associated with hearing loss.

Therefore, it was predicted that the variant does not lead to a distinct decrease in structural function, resulting in relatively mild hearing loss in patients with the p.[R143W];[V37I] genotype. From the above findings, not only the distance from the ion pore but also the characteristics of the variants, including physical–chemical changes, should be considered when investigating the effect of these variants.

In in vitro studies, the R143W variant was localized in the region of cell–cell contact and may have formed functional gap junction channels with a value of conductance similar to that of wild-type GJB2 [7]. In addition, the coexpression of the wild-type GJB2 and N206S variant resulted in the formation of more stable channels compared with the expression of the N206S variant alone[40]. In vitro coexpression studies of the wild-type GJB2 and subjected variants (L90P, R127H, and R143W) that were injected into Xenopus oocytes exhibited significantly low levels of conductivity compared to that of the wild type[9]. These variants are located on the transmembrane domain of the protomer, far from the inner pore region, similar to R143W or N206S. They also lead to the formation of hemichannel structures in the aforementioned in vitro expression systems. These reports and our model suggest that the structural defect caused by the R143W variant allows gap junctions to form, but causes distortion of connexon structure and reduces ion permeability.

In future research, we will quantitatively investigate the energy differences between the wild type and variants by performing more detailed molecular dynamics calculations, such as by the free energy perturbation method. This analysis would enable quantification of the destabilization of the protomer structure caused by R143W, and is expected to clarify the correlation between structural distortion and hearing level.

Limitations

The findings obtained in this study indicate that the R143W variant associated with the loss of a hydrogen bond may form an expressible hemichannel structure as a protein, which is unstable and functionally abnormal. However, more cases of patients with the R143W variant will be needed to prove our molecular pathology model, which can explain patients’ hearing level.

Conclusion

We concluded that the R143W variant, which causes structural destabilization in the structural model of GJB2, provides interpretation of the severity of the molecular pathology and can serve as an alternative to clinical data. Accumulation of structural models, in vitro experimental data, and careful evaluation of clinical data focused on genotype–phenotype correlation would provide a precise understanding of the molecular pathology of GJB2 variants. However, in order to make these molecular pathologies more authoritative, cases of patients with the same variant are needed.

Availability of data and materials

Data of hearing level of 22 patients who appeared to have two GJB2 pathogenic variants are available at Division of Hearing and Balance Research, National Institute of Sensory Organs.

References

  1. Zelante L, Gasparini P, Estivill X, Melchionda S, D’Agruma L, Govea N, Mila M, Monica MD, Lutfi J, Shohat M, Mansfield E, Delgrosso K, Rappaport E, Surrey S, Fortina P. Connexin26 mutations associated with the most common form of non-syndromic neurosensory autosomal recessive deafness (DFNB1) in Mediterraneans. Hum Mol Genet. 1997;6:1605–9.

    Article  CAS  PubMed  Google Scholar 

  2. Del Castillo FJ, Del,. Castillo I, DFNB1 non-syndromic hearing impairment: diversity of mutations and associated phenotypes. Front Mol Neurosci. 2017;10:428.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Zhao HB, Kikuchi T, Ngezahayo A, White TW. Gap junctions and cochlear homeostasis. J Membr Biol. 2006;209:177–86.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Azaiez H, Chamberlin GP, Fischer SM, Welp CL, Prasad SD, Taggart RT, del Castillo I, Van Camp G, Smith RJ. GJB2: the spectrum of deafness-causing allele variants and their phenotype. Hum Mutat. 2004;24:305–11.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Snoeckx RL, Huygen PL, Feldmann D, Marlin S, Denoyelle F, Waligora J, Mueller-Malesinska M, Pollak A, Ploski R, Murgia A, Orzan E, Castorina P, Ambrosetti U, Nowakowska-Szyrwinska E, Bal J, Wiszniewski W, Janecke AR, Nekahm-Heis D, Seeman P, Bendova O, Kenna MA, Frangulov A, Rehm HL, Tekin M, Incesulu A, Dahl HH, du Sart D, Jenkins L, Lucas D, Bitner-Glindzicz M, Avraham KB, Brownstein Z, del Castillo I, Moreno F, Blin N, Pfister M, Sziklai I, Toth T, Kelley PM, Cohn ES, Van Maldergem L, Hilbert P, Roux AF, Mondain M, Hoefsloot LH, Cremers CW, Löppönen T, Löppönen H, Parving A, Gronskov K, Schrijver I, Roberson J, Gualandi F, Martini A, Lina-Granade G, Pallares-Ruiz N, Correia C, Fialho G, Cryns K, Hilgert N, Van de Heyning P, Nishimura CJ, Smith RJ, Van Camp G. GJB2 mutations and degree of hearing loss: a multicenter study. Am J Hum Genet. 2005;77:945–57.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Minami SB, Mutai H, Nakano A, Arimoto Y, Taiji H, Morimoto N, Sakata H, Adachi N, Masuda S, Sakamoto H, Yoshida H, Tanaka F, Morita N, Sugiuchi T, Kaga K, Matsunaga T. GJB2-associated hearing loss undetected by hearing screening of newborns. Gene. 2013;532:41–5.

    Article  CAS  PubMed  Google Scholar 

  7. Wang HL, Chang WT, Li AH, Yeh TH, Wu CY, Chen MS, Huang PC. Functional analysis of connexin-26 mutants associated with hereditary recessive deafness. J Neurochem. 2003;84:735–42.

    Article  CAS  PubMed  Google Scholar 

  8. Mese G, Londin E, Mui R, Brink PR, White TW. Altered gating properties of functional Cx26 mutants associated with recessive non-syndromic hearing loss. Hum Genet. 2004;115:191–9.

    Article  CAS  PubMed  Google Scholar 

  9. Palmada M, Schmalisch K, Bohmer C, Schug N, Pfister M, Lang F, Blin N. Loss of function mutations of the GJB2 gene detected in patients with DFNB1-associated hearing impairment. Neurobiol Dis. 2006;22:112–8.

    Article  CAS  PubMed  Google Scholar 

  10. Brobby GW, Muller-Myhsok B, Horstmann RD. Connexin 26 R143W mutation associated with recessive nonsyndromic sensorineural deafness in Africa. N Engl J Med. 1998;338:548–50.

    Article  CAS  PubMed  Google Scholar 

  11. Kenneson A, Van Naarden BK, Boyle C. GJB2 (connexin 26) variants and nonsyndromic sensorineural hearing loss: a HuGE review. Genet Med. 2002;4:258–74.

    Article  CAS  PubMed  Google Scholar 

  12. Adadey SM, Manyisa N, Mnika K, de Kock C, Nembaware V, Quaye O, Amedofu GK, Awandare GA, Wonkam A. GJB2 and GJB6 mutations in non-syndromic childhood hearing impairment in Ghana. Front Genet. 2019;10:841.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Maeda S, Nakagawa S, Suga M, Yamashita E, Oshima A, Fujiyoshi Y, Tsukihara T. Structure of the connexin 26 gap junction channel at 3.5 A resolution. Nature. 2009;458:597–602.

    Article  CAS  PubMed  Google Scholar 

  14. Marlin S, Garabedian EN, Roger G, Moatti L, Matha N, Lewin P, Petit C, Denoyelle F. Connexin 26 gene mutations in congenitally deaf children: pitfalls for genetic counseling. Arch Otolaryngol Head Neck Surg. 2001;127:927–33.

    Article  CAS  PubMed  Google Scholar 

  15. Wattanasirichaigoon D, Limwongse C, Jariengprasert C, Yenchitsomanus PT, Tocharoenthanaphol C, Thongnoppakhun W, Thawil C, Charoenpipop D, Pho-iam T, Thongpradit S, Duggal P. High prevalence of V37I genetic variant in the connexin-26 (GJB2) gene among non-syndromic hearing-impaired and control Thai individuals. Clin Genet. 2004;20:452–60.

    Article  Google Scholar 

  16. Arnold K, Bordoli L, Kopp J, Schwede T. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics. 2006;22:195–201.

    Article  CAS  PubMed  Google Scholar 

  17. Kiefer F, Arnold K, Künzli M, Bordoli L, Schwede T. The SWISS-MODEL repository and associated resources. Nucleic Acids Res. 2009;37:387–92.

    Article  Google Scholar 

  18. Waterhouse A, Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, Heer FT, de Beer TAP, Rempfer C, Bordoli L, Lepore R, Schwede T. SWISS-MODEL: homology modeling of protein structures and complexes. Nucleic Acids Res. 2018;46:296–303.

    Article  Google Scholar 

  19. Bowie JU, Lüthy R, Eisenberg D. A method to identify protein sequences that fold into a known three-dimensional structure. Science. 1991;12(253):164–70.

    Article  Google Scholar 

  20. Lüthy R, Bowie JU, Eisenberg D. Assessment of protein models with three-dimensional profiles. Nature. 1992;5(356):83–5.

    Article  Google Scholar 

  21. Namba K, Mutai H, Kaneko H, Hashimoto S, Matsunaga T. In silico modeling of the pore region of a KCNQ4 missense mutant from a patient with hearing loss. BMC Res Notes. 2012;5:145.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, Tunyasuvunakool K, Bates R, Žídek A, Potapenko A, Bridgland A, Meyer C, Kohl SAA, Ballard AJ, Cowie A, Romera-Paredes B, Nikolov S, Jain R, Adler J, Back T, Petersen S, Reiman D, Clancy E, Zielinski M, Steinegger M, Pacholska M, Berghammer T, Bodenstein S, Silver D, Vinyals O, Senior AW, Kavukcuoglu K, Kohli P, Hassabis D. Highly accurate protein structure prediction with AlphaFold. Nature. 2021;596:583–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Tunyasuvunakool K, Adler J, Wu Z, Green T, Zielinski M, Žídek A, Bridgland A, Cowie A, Meyer C, Laydon A, Velankar S, Kleywegt GJ, Bateman A, Evans R, Pritzel A, Figurnov M, Ronneberger O, Bates R, Kohl SAA, Potapenko A, Ballard AJ, Romera-Paredes B, Nikolov S, Jain R, Clancy E, Reiman D, Petersen S, Senior AW, Kavukcuoglu K, Birney E, Kohli P, Jumper J, Hassabis D. Highly accurate protein structure prediction for the human proteome. Nature. 2021;596:590–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Mirdita M, Schütze K, Moriwaki Y, Heo L, Ovchinnikov S, Steinegger M. ColabFold: making protein folding accessible to all. Nat Methods. 2022;19:679–82.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE. UCSF chimera—a visualization system for exploratory research and analysis. J Comput Chem. 2004;25:1605–12.

    Article  CAS  PubMed  Google Scholar 

  26. Mazzoli MM, Van Camp G, Newton V, Giarbini N, Declau F, Parving A. Recommendations for the description of genetic and audiological data for families with nonsyndromic hereditary hearing impairment. Audiol Med. 2009;1:148–50.

    Google Scholar 

  27. Yamamoto N, Mutai H, Namba K, Morita N, Masuda S, Nishi Y, Nakano A, Masuda S, Fujioka M, Kaga K, Ogawa K, Matsunaga T. Prevalence of TECTA mutation in patients with mid-frequency sensorineural hearing loss. Orphanet J Rare Dis. 2017;12:157.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Ioannidis NM, Rothstein JH, Pejaver V, Middha S, McDonnell SK, Baheti S, Musolf A, Li Q, Holzinger E, Karyadi D, Cannon-Albright LA, Teerlink CC, Stanford JL, Isaacs WB, Xu J, Cooney KA, Lange EM, Schleutker J, Carpten JD, Powell IJ, Cussenot O, Cancel-Tassin G, Giles GG, MacInnis RJ, Maier C, Hsieh CL, Wiklund F, Catalona WJ, Foulkes WD, Mandal D, Eeles RA, Kote-Jarai Z, Bustamante CD, Schaid DJ, Hastie T, Ostrander EA, Bailey-Wilson JE, Radivojac P, Thibodeau SN, Whittemore AS, Sieh W. REVEL: an ensemble method for predicting the pathogenicity of rare missense variants. Am J Hum Genet. 2016;99:877–85.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Oza AM, DiStefano MT, Hemphill SE, Cushman BJ, Grant AR, Siegert RK, Shen J, Chapin A, Boczek NJ, Schimmenti LA, Murry JB, Hasadsri L, Nara K, Kenna M, Booth KT, Azaiez H, Griffith A, Avraham KB, Kremer H, Rehm HL, Amr SS, Abou Tayoun AN, ClinGen Hearing Loss Clinical Domain Working Group. Expert specification of the ACMG/AMP variant interpretation guidelines for genetic hearing loss. Hum Mutat. 2018;39:1593–613.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Kenna MA, Wu BL, Cotanche DA, Korf BR, Rehm HL. Connexin 26 studies in patients with sensorineural hearing loss. Arch Otolaryngol Head Neck Surg. 2001;127:1037–42.

    Article  CAS  PubMed  Google Scholar 

  31. Shen J, Oza AM, Del Castillo I, Duzkale H, Matsunaga T, Pandya A, Kang HP, Mar-Heyming R, Guha S, Moyer K, Lo C, Kenna M, Alexander JJ, Zhang Y, Hirsch Y, Luo M, Cao Y, Wai Choy K, Cheng YF, Avraham KB, Hu X, Garrido G, Moreno-Pelayo MA, Greinwald J, Zhang K, Zeng Y, Brownstein Z, Basel-Salmon L, Davidov B, Frydman M, Weiden T, Nagan N, Willis A, Hemphill SE, Grant AR, Siegert RK, DiStefano MT, Amr SS, Rehm HL, Abou Tayoun AN, ClinGen Hearing Loss Working Group. Consensus interpretation of the p.Met34Thr and p.Val37Ile variants in GJB2 by the clingen hearing loss expert panel. Genet Med. 2019;21:2442–52.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Wu BL, Lindeman N, Lip V, Adams A, Amato RS, Cox G, Irons M, Kenna M, Korf B, Raisen J, Platt O. Effectiveness of sequencing connexin 26 (GJB2) in cases of familial or sporadic childhood deafness referred for molecular diagnostic testing. Genet Med. 2002;4:279–88.

    Article  CAS  PubMed  Google Scholar 

  33. Bissantz C, Kuhn B, Stahl M. A medicinal chemist’s guide to molecular interactions. J Med Chem. 2010;53:5061–84.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Sakata A, Kashio A, Koyama M, Urata S, Koyama H, Yamasoba T. Hearing and hearing loss progression in patients with GJB2 Gene mutations: a long-term follow-up. Int J Mol Sci. 2023;24(23):16763.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Orzan E, Polli R, Martella M, Vinanzi C, Leonardi M, Murgia A. Molecular genetics applied to clinical practice: the Cx26 hearing impairment. Br J Audiol. 1999;33:291–5.

    Article  CAS  PubMed  Google Scholar 

  36. Schrijver I, Chang KW. Two patients with the V37I/235delC genotype: are radiographic cochlear anomalies part of the phenotype? Int J Pediatr Otorhinolaryngol. 2006;70:2109–13.

    Article  PubMed  Google Scholar 

  37. Huang Y, Yang XL, Chen WX, Duan B, Lu P, Wang Y, Xu ZM. Prevalence of p.V37I variant of GJB2 among Chinese infants with mild or moderate hearing loss. Int J Clin Exp Med. 2015;8:21674–8.

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Du Y, Huang L, Cheng X, Zhao L, Ruan Y, Ni T. Analysis of p.V37I compound heterozygous mutations in the GJB2 gene in Chinese infants and young children. Biosci Trends. 2016;10:220–6.

    Article  CAS  PubMed  Google Scholar 

  39. Tsai CY, Lu YC, Chan YH, Radhakrishnan N, Chang YY, Lin SW, Liu TC, Hsu CJ, Chen PL, Yang LW, Wu CC. Simulation-predicted and -explained inheritance model of pathogenicity confirmed by transgenic mice models. Comput Struct Biotechnol J. 2023;21:5698–711.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Ambrosi C, Walker AE, Depriest AD, Cone AC, Lu C, Badger J, Skerrett IM, Sosinsky GE. Analysis of trafficking, stability and function of human connexin 26 gap junction channels with deafness-causing mutations in the fourth transmembrane helix. PLoS ONE. 2013;8:e70916.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We are grateful to the families of the patients who participated in this study.

Funding

This work was supported by the Japan Society for Promotion of Sciences [17K11350] to HK, [18K09365] to KN, and by a Grant-in-Aid for Clinical Research from the National Hospital Organization of Japan [H30-NHO(kankakuki)-01] to TM. These funding sources were not involved in the study design; collection, analysis and interpretation of data; writing of the report; nor decision to submit the article.

Author information

Authors and Affiliations

Authors

Contributions

KN and HK designed the study and worked on structural modeling. KN and HM contributed to data analysis, curation, and writing the draft of the manuscript. TM and HM contributed to genetic analysis and evaluation. TM collected patient data, and HK finalized the manuscript.

Corresponding author

Correspondence to Hiroki Kaneko.

Ethics declarations

Ethics approval and consent to participate

This study was approved by the institutional ethics review board of the National Hospital Organization Tokyo Medical Center (Application number R20-184). Written informed consent was obtained from every patient with sensorineural hearing loss or from their parents when the patients were children.

Consent for publication

Not applicable.

Competing interests

The authors declare no conflict of interest associated with this manuscript.

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Supplementary Information

Additional file 1: Fig. S1.

Comparison of the hydrophobic environment between the wild type and V37I variant.

Additional file 2: Fig. S2.

Comparison between wild type crystal structure (gold) and variant models (R143W (cyan), N206S (light green) and N206T (light gray)) predicted by AlphaFold2.

Additional file 3: Table S1:

Clinical data of patients with each genotype.

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Namba, K., Mutai, H., Matsunaga, T. et al. Structural basis for pathogenic variants of GJB2 and hearing levels of patients with hearing loss. BMC Res Notes 17, 131 (2024). https://doi.org/10.1186/s13104-024-06793-w

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